Fig 2: Tbr1+/- mice show AMPAR and NMDAR hypofunction at amygdalar thalamic-LA synapses, which are rescued by high dietary zinc. A Schematic of electrode placement in the LA for all electrophysiology experiments. LA: lateral amygdala; BA: basal amygdala. B Representative traces of half-maximal AMPAR-mediated EPSCs recorded from WT mice and heterozygous mutant Tbr1+/- mice fed with normal or high zinc diet. C Significantly reduced half-maximal amplitude of AMPAR-mediated EPSCs in Tbr1+/- thalamic-LA synapses are restored by high dietary zinc. (Sample size, neurons/animals: WT30ppm = 13/6, Tbr+/-30 ppm = 19/8, WT150ppm = 13/5, Tbr+/-150 ppm = 12/6). D Representative traces of AMPAR- and NMDAR-mediated EPSCs. E, F Tbr1+/- mice display significantly impaired NMDAR function, measured by both reduced average amplitudes of evoked NMDAR-mediated EPSCs and reduced NMDAR/AMPAR ratio, which is prevented by dietary zinc supplementation. E, F Sample size, neurons/animals: WT30ppm = 14/6, Tbr+/-30 ppm = 14/6, WT150ppm = 10/5, Tbr+/-150 ppm = 11/5). Each point represents data from individual neurons recorded in each experimental group. All data represent mean ± standard error of the mean, analysed using one-way analysis of variance with Tukey’s post hoc test. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig 3: Disruption of mGluR5/Homer coupling alters synaptic NMDAR function and plasticity. (a) NMDAR-mediated excitatory post-synaptic currents (EPSCsNMDA) were recorded from CA1 pyramidal neurons. Scale bar = 100 µm (b) Left: representative EPSCsNMDA traces from WT, Fmr1 KO, WT treated with either TAT-mGluR5ct or TAT-mGluR5mu (both 5 µM, 4 h). Histograms: EPSCsNMDA amplitude (F(3, 35) = 6.26; **P = 0.0016; one-way ANOVA with Tukey’s Multiple Comparison). Compared to WT (175.7 ± 21.9 pA, n = 11 neurons from 7 animals), EPSCsNMDA were lower in Fmr1 KO (46.4 ± 8.4 pA, n = 7 neurons from 4 animals, **P < 0.01) and in WT treated with TAT-mGluR5ct (88.1 ± 12.7 pA, n = 13 neurons from 7 animals, *P < 0.05) but not with TAT-mGluR5mu (125.9 ± 16.8 pA n = 8 neurons from 3 animals, P = 0.14). (c) NMDA/AMPA ratio in WT, Fmr1 KO, WT treated with TAT-mGluR5ct or TAT-mGluR5mu (F(3, 32) = 4.1; *P = 0.013; one-way ANOVA with Tukey’s Multiple Comparison). NMDA/AMPA ratio (in WT 1.37 ± 0.17, n = 9 neurons from 4 animals) was reduced in Fmr1 KO (0.86 ± 0.05, n = 8 neurons from 3 animals, *P = 0.02) and in WT treated with TAT-mGluR5ct (0.88 ± 0.10, n = 9 neurons from 3 animals, *P = 0.02). (d, f) The mGluR1/5 agonist DHPG (100 µM, 5 min) induced long-term depression (mGluR-LTD) of EPSCsNMDA in WT (EPSCsNMDA amplitude: 24.9 ± 2 % of baseline, n = 8 neurons from 5 animals) but not in Fmr1 KO (EPSCsNMDA 107.8 ± 25 %, n = 6 neurons from 4 animals, **P < 0.01, WT vs. Fmr1 KO). (e, f) mGluR-LTD of EPSCsNMDA was abolished in WT treated with TAT-mGluR5ct (EPSCsNMDA 100.6 ± 15 %, n = 8 neurons from 7 animals, **P < 0.01, WT vs. TAT-mGluR5ct) but not with TAT-mGluR5mu (EPSCsNMDA 56.6 ± 11 %, n = 7 neurons from 3 animals). (f) mGluR-LTD magnitude in all conditions (F(3, 19) = 7.27, **P = 0.0019, one-way ANOVA with Tukey’s Multiple Comparison)

Fig 4: tPA-induced potentiation of NMDAR signaling is dependent of the LBS of the tPA kringle 2 domain (kringle 2-tPALBS). (a) Calcium video imaging performed on HEK-293 cells transiently transfected with GluN1-1b WT and GluN2A in combination with tPA WT or a tPA containing a point mutation within its LBS-containing kringle 2 domain (W254, tPA K2*).35 After control NMDA stimulations (used as baseline), transfected HEK-293 cells with either GluN1-1b/GluN2A/tPA WT (n=44 cells) or GluN1-1b/GluN2A/tPA K2* (n=31 cells) were incubated for 20 min with Glunomab (10 µg/ml), before a second set of NMDA stimulations. (b) Percentages of inhibition after incubation with Glunomab were calculated for each individual cell and reported as the percentages of inhibition for each group (mean±S.E.M.; N=3 independent experiments; **P<0.01 Kruskal–Wallis and Mann–Whitney tests for group comparison; #P<0.01 Wilcoxon signed-rank test for the comparison pre- and post-incubation responses). (c) Percentages of potentiated, not affected and inhibited cells

Fig 5: The callosal somatosensory innervation pattern was disrupted in Emx1cre/+; Grin1fl/fl mice.(A–E) Postnatal development of callosal projection in S1. (A) EGFP plasmid injected into lateral ventricle of embryo at embryonic day 15.5 (E15.5) and electrical pulse given to enable the plasmid to enter cortical progenitor cells of layer II/III in the ventricular zone. (B, B’) At postnatal day 5 (P5), the callosal axons from S1 had reached the white matter underneath contralateral S1. (C, C’) At P8, the callosal axons were diffusely distributed in contralateral S1. (D, D’) By P12, pruning of excess projections led to a refined innervation pattern with a narrow band limited to the S1/S2 border. (E, E’) After P12, the pattern was stable as observed at P30. (F) In P14 control mice (Emx1cre/+; Grin1fl/wt), the callosal innervation pattern of S1 of the contralateral cortex is well differentiated with a dense innervation at S1/S2 border. The pattern persists to P30 (J). (G) In GluN1 KO mice (Emx1cre/+; Grin1fl/fl), the innervation pattern was disrupted and projections were extremely diffused which also persisted to P30 (K). (H) Quantification of fluorescent intensity across the medial to lateral extent of the S1. (I, L) Quantification of fluorescence density of S1 region of control vs. GluN1 KO mice at P14 (I, p=0.002) and P30 (L, p=0.0003). Scale bar: 500 µm for all images. S1: primary somatosensory cortex; S2: secondary somatosensory cortex.